ABSTRACT A better understanding of the interplay between innate and adaptive immune responses to hepatitis B virus (HBV) in human immunodeficiency virus (HIV-1)-induced immune system impairment is crucial for the development of new antiviral immune therapeutics. Mice that carry human immune system and are affected by HIV-1 have great promise in in vivo experimental models for studying HBV/HIV co-infection. Despite current progress in humanized mouse models, species differences preclude efficient ?collaboration? of the human immune system in a shell of mouse non-immune cells. We propose to create a new model that possesses the most crucial set of human genes for lymphoid tissue development, namely the lymphotoxin beta receptor (hLT?R), two chemokines involved in T- and B-cell zone establishment (hCCL21, hCXCL13), and a thymic stromal lymphopoietin (hTSLP). In this application, we propose replacing mouse genes with hLT?R, hCCL21, hCXCL13, and hTSLP to develop a new model suitable for future HBV/HIV coinfection studies. We will employ Easi-CRISPR gene editing, our recently developed and highly robust method, to create the proposed mouse model(s). In this strain, we expect efficient generation of human follicular helper cells, which are a key component for the establishment of adaptive immunity to HBV. The ability to study the immune response to HBV in the presence of HIV-1 infection will be a new step in modeling HBV/HIV coinfection. All genetic manipulations will be performed on a strain of mice that already has human IL-34 transgene on a NOG background. The rational for NOG-hIL34 transgenic mice is that it efficiently supports human tissue-resident macrophages (including Kupffer cells) and introduces lymphoid tissue organizing human factors, which will provide a strong support for development of follicular helper T cells and the adaptive immune response to HBV antigens. We will validate the ability of the new humanized strain to control HIV-1 infection and the effects of infection on immune responses to HBsAg. Such a model will also provide a means for the development of therapeutics and address pathobiological paradigms of HBV/HIV coinfection previously not possible to study in vivo. Our immediate goal is to improve the existing model to create a new, invaluable tool for researchers that can support a wide range of HBV/HIV coinfection studies. By testing the efficacy of human immune system development and function in all four modifications, we can determine whether to combine all genotypes into one mouse strain. As the next step in development, we will introduce TK-NOG background for efficient transplanting of human hepatocytes. Our long-term goal is to facilitate studies of innate and adaptive immunity in HBV/HIV-1 coinfection.